Treatment of radiation disorders

ABSTRACT

The present invention provides methods and compositions for the treatment of DNA damage related disorders. One embodiment is a method for the inhibition of side effects associated with chemotherapeutic and radiotherapeutic agents using chloroquine compounds. Another embodiment is a method for treatment and/or prevention of lethal or sub-lethal radiation toxicities associated with terrorist acts or war.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. ______, filed on May 26, 2004, Attorney Docket No.29202-710.831, which is a national stage application under 35 USC 371 ofPCT/US03/37838 filed Nov. 26, 2003 which claims the benefit of priorityfrom U.S. patent application Ser. No. 10/351,733 filed Jan. 24, 2003 andSer. No. 10/307,077, filed Nov. 27, 2002 which are incorporated hereinby reference in their entirety.

This invention was made in the course of research sponsored by theNational Institutes of Health (NIH Grant Nos. CA71387). The U.S.government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Cancer is now the second leading cause of death in the United States.Over 1 million new cases of cancer are expected to be diagnosed in 2003and over 500,000 people are expected to die of cancer.

Cancer is typically treated with one or a combination of three types oftherapies: surgery, radiation, and chemotherapy. Overall costs forcancer, including treatments, were approximately $170 billion dollars in2002. The cancer treatments are not only expensive; they are ineffectivemost of the time and also have many side effects. Hence, there is ademand for more effective cancer prevention and treatment agents, aswell as for the prevention and treatment of DNA damage relatedconditions.

Radiation is all around us. Exposure to radiation includes radiationfrom natural sources and man-made sources. Acute and/or chronic exposureto radiation causes several adverse effects, including occasionallycausing death. The current treatments for treating the adverse effectsfollowing exposure to radiation are ineffective most of the time andalso have many side effects. Hence, there is a demand for more effectivetreatment of effects following exposure to radiation.

SUMMARY OF THE INVENTION

The present invention provides compositions, methods, and kits for thetreatment and/or prevention of DNA damage related disorders, disorderscaused by radiation exposure, and death associated with radiationexposure. In one embodiment, a chloroquine compound is administered forthe prevention of DNA damage related disorders, like cancer andradiation related disorders. In another embodiment, the chloroquinecompound does not prevent a localized skin cancer alone. In yet anotherembodiment, the cancers prevented by the chloroquine compounds are alocalized skin cancer and a cancer that is not a localized skin cancer.Also described herein are methods of inhibiting the side effects ofchemotherapeutic and/or radiotherapeutic agents using chloroquinecompounds. Another embodiment is the prevention and/or treatment oflethal or sub-lethal radiation toxicities associated with acts ofterrorism or war on response to such acts. The present invention alsoprovides compositions, methods, and kits for use in the treatment ofradiation related disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Kaplan-Meier survival curve of C57/BL6 mice afterexposure to 8 Gy total body irradiation (TBI). Half of the cohortreceived a dose of chloroquine (dashed line) by either i.p. injection(1.75 mg/kg or 3.5 mg/kg) or in their drinking water (3.5 mg/kg or 7mg/kg) the day before the TBI. The one mouse which died in thechloroquine-treated group received 1.75 mg/kg by i.p. injection.

FIG. 2 shows that chloroquine treatment enhances survival after TBI byenhancing recovery of hematopoietic progenitor cells. Five mice received3.5 mg/kg chloroquine (C) by i.p. injection 24 and 4 hours prior to TBI(bars with diagonal stripes). Five mice received no chloroquine(stippled bars). Fourteen days after irradiation, the cellularity (openbars) of hematopoietic tissues (spleen, thymus, bone marrow) wasassessed by a blinded observer on a scale of 0-3 with 3 being normalcellularity. The bars represent the average cellularity of the tissuesfrom the 5 mice in each group.

FIG. 3 shows a Kaplan-Meier survival curve of AT mice after exposure to8 Gy TBI. Half of the cohort received a dose of 3.5 mg/kg chloroquine(CHL; dashed line) by i.p. injection 24 and 4 hours prior to the TBI.

FIG. 4 demonstrates that chloroquine treatment prevents the developmentof tumors in Eμ-myc mice. After weaning, a cohort of transgenic miceexpressing the c-myc oncogene were started on chloroquine (CHL) at 7.0mg/kg in the drinking water ((+), solid line). Within 100 days, all ofthe mice with no drug in the water had died of leukemia, while none ofthe cohort of mice on drug had succumbed. The latter group of mice wasthen divided into two groups (timing of this event depicted by heavyarrow), one group of which was taken off of chloroquine ((−), dashedline) and the other group of which was started on i.p. injections of 3.5mg/kg of chloroquine once a week. Within a month, all of the mice takenoff of chloroquine had developed malignancies and all of the mice on theweekly i.p. injections remained tumor-free for months.

FIG. 5 illustrates that chloroquine treatment reduces the development oftumors in mice injected with the potent chemical carcinogen,3-methylcholanthrene (3-MC). Chloroquine (CHL, 3.5 mg/kg) was given byi.p. injection 24 and 4 hours prior to 3-MC injection in 30 mice and 30mice received the carcinogen with no chloroquine pretreatment. Thepercentage of animals remaining tumor-free is plotted. Statisticalsignificance, log rank test P<0.0001.

FIG. 6 demonstrates that chloroquine treatment reduces the developmentof tumors in mice exposed to ionizing radiation in a protocol thatinduces thymic lymphomas. Chloroquine (CHL, 3.5 mg/kg) was given by i.p.injection 24 and 4 hours prior to irradiation in four successive weeksand animals were subsequently observed for the development of tumors.Statistical significance, log rank test P=0.0012.

FIG. 7 shows tumor incidence in wildtype mice receiving either placeboor CHQ before 3-MC injection. CHQ markedly protects from tumordevelopment.

FIG. 8 shows tumor incidence in ATM-null mice receiving either placeboor CHQ before 3 MC injection. CHQ does not protect from tumordevelopment.

FIG. 9 shows tumor incidence in p53-null mice receiving either placeboor CHQ before 3 MC injection. CHQ does not protect from tumordevelopment.

FIG. 10 demonstrates the efficacy of two chloroquine compounds inpreventing, in varying degree, the change in coat color in mice treatedwith 8 GY radiation.

DETAILED DESCRIPTION OF THE INVENTION

Chloroquine Compounds

The present invention provides methods, compositions, and kits for theprevention and/or treatment of DNA damage related disorders and deathdue to radiation exposure. Chloroquine compounds are useful inpracticing the invention described herein. The term “chloroquinecompounds” as used herein means chloroquine-like compounds, chloroquineand enantiomers, analogs, derivatives, metabolites, pharmaceuticallyacceptable salts, and mixtures thereof. Examples of chloroquinecompounds include, but are not limited to, chloroquine phosphate,hydroxychloroquine, chloroquine diphosphate, chloroquine sulphate,hydroxychloroquine sulphate, and enantiomers, analogs, derivatives,metabolites, pharmaceutically acceptable salts, and mixtures thereof.The term “chloroquine-like compounds” as used herein means compoundsthat mimic chloroquine's biological and/or chemical properties.

In a specific embodiment, the invention is practiced with chloroquine.The chemical structure of chloroquine,N⁴-(7-Chloro-4-quinolinyl)-N¹,N¹-diethyl-1,4-pentanediamine or7-chloro-4-(4-diethylamino-1-methylbutylamino) quinoline, is as follows:

Chloroquine (The Merck Index, p. 2220, 1996) is a syntheticallymanufactured drug containing a quinoline nucleus. Suitable synthesistechniques for chloroquine are well known in the art. For example seeU.S. Pat. No. 2,233,970.

As mentioned above, the chloroquine compounds useful herein includechloroquine analogs and derivatives. A number of chloroquine analogs andderivatives are well known. For example, suitable compounds and methodsfor synthesizing the same are described in U.S. Pat. Nos. 6,417,177;6,127,111; 5,639,737; 5,624,938; 5,736,557; 5,596,002; 5,948,791;5,510,356; 2,653,940; 2,233,970; 5,668,149; 5,639,761; 4,431,807; and4,421,920. In certain preferred embodiments, chloroquine is used in themethods described herein. In other embodiments hydroxychloroquine isused.

Examples of suitable chloroquine compounds include chloroquinephosphate; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline(desmethylchloroquine);7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinol ine(hydroxychloroquine);7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;hydroxychloroquine phosphate;7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline(desmethylhydroxychloroquine);7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino]-6-methoxydihydrochloride quinoline;1-acetyl-1,2,3,4-tetrahydroquinoline;8-[4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-butyryl-1,2,3,4-tetrahydroquinoline;7-chloro-2-(o-chlorostyryl)-4-[4-diethylamino-1-methylbutyl]aminoquiinoline phosphate; 3-chloro-4-(4-hydroxy-.alpha.,.alpha.′-bis(2-methyl-1-pyrrolidinyl)-2,5-xyl idinoquinoline,4-[(4-diethylamino)-1-methylbutyl)amino]-6-methoxyquinoline;3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde;1,1′-pentamethylenediquinoleinium diiodide; and 8-quinolinol sulfate,enantiomers thereof, as well as suitable pharmaceutical salts thereof.

Additional suitable chloroquine derivatives include aminoquinolinederivatives and their pharmaceutically acceptable salts such as thosedescribed in U.S. Pat. Nos. 5,948,791 and 5,596,002. Suitable examplesinclude(S)—N₂-(7-Chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine;(R)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-2,N₂,N₂-trimethyl-propane-1,2-diamine;N₃-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,3-diamine;(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-piperidin-3-yl)-amine;(RS)-(7-choro-quinolin-4-yl)-(1-methyl-pyrrolidin-3-yl)-amine;(RS)—N₂-(7-Chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,2-diamine;(RS)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine;(S)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine;(R)—N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-propane-1,2-diamine;(RS)-7-chloro-quinolin-4-yl)-(1-methyl-2-pyrrolidin-1-yl-ethyl)-am ine;N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-ethane-1,2-diamine;N₂-(7-chloro-quinolin-4-yl)-N₁,N₁-diethyl-ethane-1,2-diamine;N₃-(7-chloro-quinolin-4-yl)-N₁,N₁-dimethyl-propane-1,3-diamine;(R)—N₁-(7-chloro-quinolin-4-yl)-N₂,N₂-dimethyl-propane-1,2-diamine;(S)—N₁-(7-chloro-quinoline-4-yl)-N₂,N₂-dimethyl-propane-1,2-diamine;(RS)-(7-chloro-quinolin-4-yl)-(1-methyl-pyrrol idin-2-yl-methyl)-amine;N₁-(7-Chloro-quinolin-4-yl)-N₂-(3-chloro-benzyl)-2-methyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-N₂-(benzyl)-2-methyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-N₂-(2-hydroxy-3-methoxy-benzyl)-2-methyl-propane-1,2-diamine;N₁-(7-chloro-quinolin-4-yl)-N₂-(2-hydroxy-5-methoxy-benzyl)-2-methyl-propane-1,2-diamine;andN₁-(7-chloro-quinolin-4-yl)-N₂-(4-hydroxy-3-methoxy-benzyl)-2-methyl-propane-1,2-diamine;(1S,2S)-N₁-(7-chloro-quinolin-4-yl)-N₂-(benzyl)-cyclohexane-1,2-diamine;(1S,2S)-N₁-(7-chloro-quinolin-4-yl)-N₂-(4-chlorobenzyl)-cyclohexane-1,2-diamine;(1S,2S)-N₁-(7-chloro-quinolin-4-yl)-N₂-(4-dimethylamino-benzyl)-cyclohexane-1,2-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N-4-(benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(3-chloro-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(2-hydroxy-4-methoxy-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(3,5-dimethoxy-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-methylsulphanyl-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-diethylamino-benzyl)-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(biphenyl-4-yl)methyl-cyclohexane-1,4-diamine;trans-N₁-(7-chloro-quinolin-4-yl)-N₄-[2-(3,5-dimethoxy-phenyl)-ethyl]-cyclohexane-1,4-diamine;cis-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-methoxy-benzyl)-cyclohexane-1,4-diamine;trans-N₁-(7-chloro-quinolin-4-yl)-N₄-(4-dimethylamino-benzyl)-cyclohexane-1,4-diamine;andtrans-N₁-(7-chloro-quinolin-4-yl)-N₄-(2,6-difluoro-benzyl)-cyclohexane-1,4-diamine.

Chloroquine compounds such as chloroquine may exhibit the phenomena oftautomerism, conformational isomerism, geometric isomerism, and/oroptical isomerism. The invention covers any tautomeric, conformationalisomeric, optical isomeric and/or geometric isomeric forms of thechloroquine compounds, as well as mixtures of these various differentforms.

Chloroquine and hydroxychloroquine are generally racemic mixtures of(−)- and (+)-enantiomers. The (−)-enantiomers are also known as(R)-enantiomers (physical rotation) and 1-enantiomers (opticalrotation). The (+)-enantiomers are also known as (S)-enantiomers(physical rotation) and r-enantiomers (optical rotation). The metabolismof the (+)- and the (−)-enantiomers of chloroquine are described inAugustijins and Verbeke (1993) Clin. Pharmacokin. 24(3):259-69;Augustijins, et al. (1999) Eur. J. Drug Metabol. Pharmacokin.24(1):105-8; DuCharme and Farinotti (1996) Clin. Pharmacokin.31(4):257-74; Ducharme, et al. (1995) Br. J. Clin. Pharmacol.40(2):127-33. Preferably, the (−)-enantiomer of chloroquine is used. Inother embodiments, the (+) enantiomer of chloroquine is employed.

In certain embodiments, essentially pure (+) or (−) enantiomers are usedin the methods described herein. The term “essentially pure” does notrequire a 100% enantiomer. The term refers to about more than 95% pureenatiomer, preferably more than about 90%, more preferably more thanabout 85% pure, even more preferably more than about 80%, and mostpreferably more than 75% pure. In certain embodiments mixturescontaining various ratios of the (+) or (−) form in combination with theracemic mixture are used. In certain embodiments employing the (+) or(−) forms, the doses of the enantiomer used can be less than if theracemic mixture were used. The enantiomers of chloroquine andhydroxychloroquine may be prepared by procedures known to the art.

The chloroquine compounds may metabolize to produce active metabolites.The used of active metabolites is also within the scope of the presentinvention.

Not intending to be limited by one mechanism, it is believed that thechloroquine compounds and chloroquine-like compounds act by enhancingthe activity of Ataxia-Telangiectasia Mutated (ATM) kinase anddownstream events. The agonistic properties of chloroquine on ATM kinasehave been demonstrated. Hence, it is intended herein thatchoroquine-like compounds include compounds that are agonists of ATMkinase. Agonists of ATM kinase include compounds that promote thedissociation of ATM into active monomers and/or compounds that promotephosphorylation of a serine corresponding to the residue 1981 of ATMkinase of SEQ ID NO:1.

Use of Chloroquine Compounds

In one aspect, the invention provides methods of treating an animalsubject, including a human. The term “animal subject” as used hereinincludes humans as well as other mammals. The methods described hereingenerally involve the administration of effective amounts of chloroquinecompounds and/or chloroquine like compounds for the treatment and/oreprevention of DNA damage related disorders. The term “DNA damage relateddisorders” include, but are not limited to, cancer, aging, disorderscaused by damage to DNA due to exposure to carcinogens, toxins, freeradicals, like oxygen radical, or DNA damaging radiations like ionizingradiation and UV radiation. The chloroquine compounds are also usefulfor prevention of tissue injury resulting from ischemia, such as thatwhich occurs following myocardial infarction or stroke. The effects ofthe chloroquine compounds used in the methods described herein includesystemic, local, and topical effects. It is preferred that the effectsof the chloroquine compounds in the methods described herein aresystemic.

In one embodiment, the chloroquine compounds are used as prophylacticsto prevent DNA damage related disorders. The chloroquine compounds areuseful in the prevention of cancers caused by toxins, carcinogens, DNAdamaging radiations, and/or genetic mutations. For example, chloroquinecompounds are useful in the prevention of cancers caused by exposure totoxins and carcinogens like aromatic hydrocarbons, cigarette smoke,acetyl amino fluorine, MTBE, etc. Also, chloroquine compounds are usefulin prevention of cancers caused by DNA damaging radiations like UV andionizing radiation. The ionizing radiations includes both natural andtherapeutic radiation exposures. Examples of ionizing radiations areX-rays for diagnostics and radiation therapy used for tumors andunintended exposure to radiation as an act of terrorism or war.

The prophylactic uses for cancer described herein are not envisioned toencompass the prevention solely of localized skin carcinomas like basalcell epithelioma and squamous cell carcinoma, skin carcinomas, Burkitt'slymphoma, or skin pathologies caused by harmful radiation. When used inpatients with actinic keratosis, it is envisioned the chloroquinecompounds do not solely inhibit basal cell epithelioma and squamous cellcarcinoma. In one embodiment, the chloroquine compounds are used toprevent a localized skin carcinoma and at least one cancer that is not alocalized skin carcinoma. Examples of cancers that are not localizedskin carcinomas include, but are not limited to, melanomas, lymphomas,prostate cancer, breast cancer, colon cancer, lung cancer,retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.

In a preferred embodiment, chloroquine compounds are used in theprevention of one or more of the following cancers—melanomas, prostatecancer, breast cancer, colon cancer, lung cancer, non-Hodgkins lymphoma,retinoblastoma, neuroblastoma, sarcomas, and ovarian cancer.

The chloroquine compounds can be used to prevent secondary cancers,i.e., cancers that are caused by radiation therapy and chemotherapy usedto treat the primary cancer. In one embodiment, the chloroquinecompounds are used to prevent the occurrence of breast cancer inpatients receiving radiation therapy for non-Hodgkin's lymphoma. Also,in these patients the chloroquine compounds can be used to inhibit thecellular damage caused by the radiation therapy to normal cells andenhance the repair process of the normal cells. The chloroquinecompounds are also suitable for prevention of the reoccurrence ofcancers in patients who have had prior incidences of cancer.

In one embodiment, the chloroquine compounds are administered todecrease or prevent the side-effects of radiation therapy used to treatcancer. The chloroquine compounds can be administered prior to, during,or after treatment with radiation. In this embodiment, the beneficialeffect of the chloroquine compounds is contemplated to be not solelylimited to a beneficial effect on pathological skin conditions like skincarcinomas and dermatoses. The use of chloroquine compounds incombination with radiation therapy is contemplated to protect the normalcells and inhibit the cellular damage caused by the radiation therapy tonormal cells and enhance the repair process of the normal cells.

In one embodiment, the chloroquine compounds are used inimmunosuppressed patients, like transplant patients. In immunosuppressedpatients, the chloroquine compounds can be used to prevent cancers. Thechloroquine compounds can be used to prevent Epstein Barr virus inducedlymphoproliferative syndrome.

In another embodiment, chloroquine compounds are used as prophylacticsto inhibit side effects of frequent exposure to X-rays in athletes. Thismethod would also be useful for other patient populations that arefrequently exposed to DNA damaging radiations, such as X-raytechnicians, pilots, police officers, astronauts, and the like. It isknown that exposure to X-rays causes DNA damage. Administration ofchloroquine compounds is contemplated to inhibit the side-effects offrequent exposure to DNA damaging radiations, including inhibiting thedamage to cells due to damage to DNA.

The present invention also provides methods for preventing DNA damage,inhibiting the effects of DNA damage, and stimulating cellular responseto DNA damage by administering an effective amount of chloroquinecompounds. Not intending to be limited by one mechanism of action, it iscontemplated that cellular responses are enhanced by an agonisticactivity on ATM kinase by priming the cell to respond to agents whichcause DNA damage. Further details on ATM kinase are provided inInternational PCT application no. US03/38091 filed Nov. 26, 2003, whichis incorporated by reference herein in its entirety.

The prophylactic benefits of chloroquine compounds can be obtained byadministering in advance of exposure to the DNA damaging agent toprovide the enhancing effect in one embodiment. The amount of time priorto the exposure to the DNA damaging agent that the chloroquine compoundis administered can vary from days, hours, to minutes. Also, thechloroquine compounds can be administered during exposure to the DNAdamaging agent or after such exposure. In one embodiment, the effectiveamount of a chloroquine compound is an amount which reduces DNA damage,reduces DNA mutation or increases survival of cells exposed to a DNAdamaging agent when compared to cells exposed to the same DNA damagingagent and not receiving a chloroquine compound. In another embodiment,the effective amount of a chloroquine compound is an amount whichproduces anti-oxidant effects.

The prophylactic use of chloroquine includes the prevention of tissueinjury resulting from ischemia, such as that which occurs followingmyocardial infarction or stroke. While not intending to be limited toone mechanism of action, it is believed that the chloroquine compoundsprevent cellular death due to oxidative damage during reperfusion and assuch can ameliorate tissue injury resulting from ischemic injury.

In one embodiment, chloroquine compounds are used in the treatment ofDNA damage related disorders. The chloroquine compounds are usedpreferably in combination with chemotherapeutic or radiotherapeuticagents to prevent the side-effects associated with the chemotherapeuticagents. It is known that chloroquine compounds can inhibit multiple drugresistance. Hence, it is not intended that the methods described hereinproduce a beneficial effect on multiple drug resistance alone. In apreferred embodiment, the beneficial effects of chloroquine compounds,when used in combination with chemotherapeutic agents, are due tomodulation of ATM kinase activity. It is contemplated that thechloroquine compounds protect the normal cells and inhibit the cellulardamage caused by the radiation therapy to normal cells and enhance therepair process of the normal cells.

In one embodiment, the chloroquine compounds are used to treat and/orprevent disorders caused by oxidative damage. The chloroquine compoundscan be administered with anti-oxidants, like vitamin B12, to stimulatethe cellular response to DNA damage and promote the repair of the cellsexposed to the oxidative agents.

Treatment of Radiation Therapy Related Adverse Effects

In one aspect of the invention the chloroquine compounds are used in thetreatment of adverse effects associated with radiation therapy. Thechloroquine compounds may be used to therapeutically treat the adverseeffects of radiation therapy and/or prophylactically to prevent theoccurrence of the adverse effects associated with radiation therapy. Inpreferred embodiments, the use of the chloroquine compounds does notadversely affect the efficacy and/or potency of the radiation therapy inthe disease being treated.

Radiation therapy, also known as radiotherapy, x-ray therapy, orirradiation, is the treatment of disease using penetrating beams ofhigh-energy or low-energy waves or streams of particles calledradiation. The radiation is used for the treatment of cancer and isusually administered from special machines or from radioactivesubstances. The doses of radiation that damage or destroy the diseasedcells, such as cancer cells, can also injure or kill normal cells. Theseeffects of radiation on normal cells cause treatment side effects. Inone embodiment, the chloroquine compounds described herein are used inthe treatment of adverse effects associated with radiation therapy. Thechloroquine compounds can be administered prior to the radiation therapyor after the radiation therapy is started. Preferably the chloroquinecompounds minimize the effects of the radiation therapy on normal cells.

The high energy rays used for radiation therapy can include for example,x-rays, an electron beam, or cobalt-60 gamma rays. Also beams of protonsor neutrons may be used for radiation therapy. Internal radiationtherapy places the radiation source as close as possible to the diseasedcells. Some of the radioactive substances used for internal radiationtreatment include cesium, iridium, iodine, phosphorus, and palladium.

Side effects of treatment with radiation include temporary or permanentloss of hair in the area being treated, skin irritation, temporarychange in skin color in the treated area, and tiredness. Some people whoreceive radiation to the head and neck experience redness and irritationin the mouth, a dry mouth, difficulty in swallowing, changes in taste,or nausea. Other possible side effects include a loss of taste,earaches, and swelling. Radiation therapy can cause hair loss(alopecia). Radiation therapy can also cause low white blood cell countsor low levels of platelets. Neutropenia, which refers to an abnormallylow number of neutrophils in the blood, can also be an adverse effectassociated with radiation therapy.

In certain embodiments, the chloroquine compounds are used to treat theadverse effects of radiation therapy on the skin. In other embodiments,the compounds are used to treat alopecia associated with radiationtherapy. In other preferred embodiments, the chloroquine compounds areused to treat the side effects associated with blood, such asneutropenia.

Treatment of Radiation Related Disorders

In one aspect of the invention the chloroquine compounds are used in thetreatment of radiation-related disorders.

Radiation is typically classified as non-ionizing and ionizingradiation. Examples of sources of non-ionizing radiation include, butare not limited to, power lines, AM/FM radio and televsion, microwaveoven, heat lamps, and tanning salons. In one embodiment, chloroquinecompounds are used to treat adverse health effects caused bynon-ionizing radiations. The compounds can be used therapeuticallyand/or prophylactically.

The kinds of ionizing radiation include alpha particles, beta particles,gamma rays, and x-rays. Ionizing radiations have enough energy to breakchemical bonds and typically cause biological damage by breaking and/ordamaging DNA bonds. In preferred embodiments, the chloroquine compoundsare used to treat adverse health effects caused by ionizing radiations.The compounds can be used therapeutically and/or prophylactically.

Typically, due to the biological effects of radiation, cells either dieor due to the damage to the DNA the cells may mutate. The mutations canbe such that the effects of the mutation are seen immediately or afterseveral days, months, or years. Also, the mutations could be passed onthe affected individuals offsprings or may show up many generationslater. In certain embodiments, the use of chloroquine has a beneficialeffect on the cell death and/or the mutations of the cells followingexposure to radiation. For example, use of chloroquine prior to exposureto radiation and/or following to radiation can decrease the number ofmutations and thus cause a decrease in the adverse genetic effectscaused by exposure to radiation.

Acute radiation syndrome (ARS) is caused by exposure of the body to highdoses of radiation usually over a short period of time. The symptomsinclude nausea, vomiting, diarrhea, loss of appetite, fatigue, fever,diminished organ function, and possibly even seizures, coma, and death.ARS also typically includes skin damage, such as swelling, itching,redness of skin, and hair loss. In some embodiments, chloroquinecompounds are used therapeutically and/or prophylactically for ARS. Inthe treatment of ARS, in some embodiments, the beneficial effects arenot solely on the skin damage caused by radiation. Preferably, thechloroquine compounds have a beneficial effects on both the skin damageand one or more of the non-skin related symptoms of ARS, such as thenausea, vomiting, diarrhea, loss of appetite, fatigue, fever, diminishedorgan function, seizures and coma.

Exposure to radiation can also cause cancers, such as leukemia, breast,bladder, colon, liver, lung, esophagus, ovarian, multiple myeloma, andstomach cancers. Other cancers that can be caused by radiation include,prostate, nasal cavity/sinuses, pharyngeal, and laryngeal, andpancreatic cancer. In some embodiments, chloroquine compounds are usedtherapeutically and/or prophylactically for cancers caused by exposureto radiation.

In some embodiments, the chloroquine compounds are used by astronauts,prior to or during space flight, to reduce the adverse effects caused byexposure to radiation during space flights. In other embodiments, themethods described herein are employed for the treatment of hospitalpersonnel who are exposed to X-ray radiation. In yet other embodiments,personnel who are exposed to radiation in war situations or have to workin areas with abnormally high levels of natural radiation are treatedwith the chloroquine compounds. Also, the compounds and methodsdescribed herein can be used to treat personnel who are involved inclean-up operations following an accidental or intentional, e.g., aterrorist attack, release of radiation.

In some embodiments, the chloroquine compounds can be used for treatmentin anticipation of or following exposure to a “dirty bomb.” A “dirtybomb” typically refers to a bomb that combines conventional explosives,such as dynamite, with radioactive materials in the form of powder orpellets. Also, the chloroquine compounds can be used in the therapeuticand/or prophylactic treatment of medical and other personnel who wouldbe involved in the treatment and clean-up operations following theexplosion of a dirty bomb. In certain embodiments, hospitals,pharmacies, and non-medical personnel stockpile chloroquine compounds tobe used in the event of an explosion of a dirty bomb. The chloroquinecompounds from such a stockpile can be used by subjects exposed to theradiation from the dirty bomb and subjects likely to exposed to theradiation.

In certain other embodiments, the chloroquine compounds are used for theprevention of death and/or for the prophylactic treatment of soldiersand other personnel entering areas with expected weapons of massdestruction or expected to encounter radiation exposure due to an act ofwar or terrorism.

In certain preferred embodiments, chloroquine and hydroxychloroquine areused in the treatment of radiation related disorders. Even morepreferred is the use of chloroquine. In other embodiments, the pureenantiomers of chloroquine or hydroxychloroquine, either the (+) or (−)form are used.

Therapeutic and Prophylactic Benefits

In one embodiment, the chloroquine compounds are used as prophylacticagents. For prophylactic benefit, the chloroquine compound may beadministered to a patient at risk of developing a DNA damage relateddisorder like cancer or to a patient reporting one or more of thephysiological symptoms of a DNA damage related disorder, even though adiagnosis of such disorder may not have been made. A prophylacticbenefit is achieved when a disorder is prevented from afflicting apatient. This prevention can include the affliction of the patient witha milder form of the disorder or the appearance of fewer or no symptomsof the disorder being prevented or the absence of the disorder in thepatient being treated.

In addition to a prophylactic benefit, the chloroquine compounds can beused for their therapeutic benefits. In one embodiment, the chloroquinecompounds are used to treat DNA damage related disorders. In a preferredembodiment, the beneficial effect of the chloroquine compounds is notdue to an inhibition of multiple drug resistance. The term “treating” asused herein includes achieving a therapeutic benefit and/or aprophylactic benefit. By therapeutic benefit is meant eradication oramelioration of the underlying disorder being treated. For example, in acancer patient, therapeutic benefit includes eradication or ameliorationof the underlying cancer. Also, a therapeutic benefit is achieved withthe eradication or amelioration of one or more of the physiologicalsymptoms associated with the underlying disorder such that animprovement is observed in the patient, notwithstanding that the patientmay still be afflicted with the underlying disorder. For example,administration of a chloroquine compound to a patient suffering fromcancer provides therapeutic benefit not only when the patient's tumormarker level is decreased, but also when an improvement is observed inthe patient with respect to other complications that accompany thecancer like pain and psychiatric disorders.

Effective Amount

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the chloroquine compoundrequired in the methods described herein. Pharmaceutical compositionssuitable for use in the present invention include compositions whereinthe chloroquine compound and other optional active ingredients arepresent in an effective amount. The effective amounts include doses thatpartially or completely achieve the desired therapeutic, prophylactic,and/or biological effect. The actual amount effective for a particularapplication will depend on the condition being treated and the route ofadministration. Determination of an effective amount is well within thecapabilities of those skilled in the art, especially in light of thedisclosure herein.

The effective amount for use in humans can be determined from animalmodels. For example, a dose for humans can be formulated to achievecirculating and/or gastrointestinal concentrations that have been foundto be effective in animals.

In one embodiment, the effective amount can include the dose ranges,modes of administration, formulations, etc., that have been recommendedor approved by any of the various regulatory or advisory organizationsin the medical or pharmaceutical arts (eg, FDA, AMA) or by themanufacturer or supplier. Effective amounts of chloroquine can be found,for example, in the Physicians Desk Reference.

The daily dosage range of chloroquine, in one embodiment, can varybetween about 0.1 mg/kg to about 2 gm/kg body weight. The daily dose ofa chloroquine compound may be less than about 2 gm/kg, less than about1.5 gm/kg, or less than about 1 gm/kg. In one embodiment, the daily doseof a chloroquine coumpound is more than about 0.5 mg/kg, more than about500 mg/kg, or more than about 1 gm/kg. Preferred daily dosage ranges ofa chloroquine compound are about 0.5 mg/kg to about 50 mg/kg or about1.0 mg/kg to about 10 mg/kg or about 30 mg/kg to about 50 mg/kg bodyweight. Preferred doses of chloroquine diphosphate andhydroxychloroquine are about 3.5 mg/kg and 7.0 mg/kg.

The dosage can vary depending on the subject being treated. For example,a preferred dosage in mice is 3.5 mg/kg once or twice a day. Theequivalent dosages in monkeys and humans are shown in the table below.Man (60 kg) Mouse (20 g) Monkey (3.0 kg) Man (60 kg) CHG Equivalent 3.5mg/kg 0.875 mg/kg  0.292 mg/kg 17.5 mg CHQ 7.0 mg/kg 1.75 mg/kg 0.583mg/kg 35.0 mg CHQ

Preferred dosages ranges in human are from 0.05-1 mg/kg, more preferably0.1 to 0.8 mg/kg, more preferably 0.2-0.6 mg/kg. In patients whose riskto cancer is occasioned by a distinct event (e.g., exposure tocarcinogen or radiation), the dosage is preferably administered dailybefore, during and/or immediately following the event, for a totalperiod of at least 1 day, 3 days, a week or a month. For example, if therisk of exposure is known in advance, an exemplary regime entailsadministering the chloroquine compound on the day before, the day ofexposure and the day after exposure. If the risk of exposure is notknown in advance, an exemplary regime entails administering thechloroquine compound at least one the day of exposure and the dayfollowing exposure. For patients subject to a chronic risk (e.g.,through genetic variation), the dosage is preferably administered weeklyfor an indefinite period. The dosage range can be lower e.g., 0.05-0.2mg/kg per day or per week of chloroquine if a purified enantiomer isused, such as the purified (−) form or the purified (+) form.

In some embodiments, the effective amount of chloroquine is administeredonce a month, every other week, once a week, more than once a week, oronce a day. The dose of chloroquine can be administered once or morethan once a day. In yet another embodiment, the effective amount of achloroquine compound is an amount that produces the intended beneficialeffects but does not produce the side-effects associated withchloroquine compounds, like retinoblastoma.

In one embodiment, the invention provides a kit comprising a chloroquinecompound packaged in association with instructions teaching a method ofusing the compound according to one or more of the above-describedmethods. The kit can contain the chloroquine compound packaged in unitdosage form.

Routes of Administration and Formulation

The compounds useful in the present invention, or pharmaceuticallyacceptable salts thereof, can be delivered to the patient using a widevariety of routes or modes of administration. Suitable routes ofadministration include, but are not limited to, inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.

The formulations useful herein can administer the chloroquine compoundstopically or systemically. In one embodiment, the formulation ofchloroquine compound is administered systemically. In anotherembodiment, the formulation of chloroquine compound has a systemiceffect if administered either topically or systemically.

The term “pharmaceutically acceptable salt” means those salts whichretain the biological effectiveness and properties of the compounds usedin the present invention, and which are not biologically or otherwiseundesirable. Such salts include salts with inorganic or organic acids,such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitricacid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid,acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid,malic acid, citric acid, tartaric acid or maleic acid. In addition, ifthe compounds used in the present invention contain a carboxy group orother acidic group, it may be converted into a pharmaceuticallyacceptable addition salt with inorganic or organic bases. Examples ofsuitable bases include sodium hydroxide, potassium hydroxide, ammonia,cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine andtriethanolamine.

If necessary, the compounds and useful herein may be administered incombination with other therapeutic agents. The choice of therapeuticagents that can be co-administered with the compounds of the inventionwill depend, in part, on the condition being treated.

Agents used in accordance with the methods of the invention may beconveniently administered in a pharmaceutical composition containing theactive compound in combination with a suitable carrier. Suchpharmaceutical compositions may be prepared by methods and containcarriers which are well-known in the art. A generally recognizedcompendium of such methods and ingredients is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippingcottWilliams & Wilkins: Philadelphia, Pa., 2000. Apharmaceutically-acceptable carrier, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, is involved in carrying or transporting the subject compoundfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient.

Examples of materials which may serve as pharmaceutically-acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;lycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in harmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Agents of use in the invention may be administered parenterally (forexample, by intravenous, intraperitoneal, subcutaneous or intramuscularinjection), topically (including buccal and sublingual), orally,intranasally, intravaginally, or rectally, with oral administrationbeing particularly preferred.

For oral therapeutic administration, the composition may be combinedwith one or more carriers and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums, foods and the like. Also, for oral consumption the activeingredient may be dissolved or suspended in water or other edible oralsolutions. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 0.1 to about 100% of the weight of a given unit dosage form. Theamount of active agent in such therapeutically useful compositions issuch that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. The above listingis merely representative and one skilled in the art could envision otherbinders, excipients, sweetening agents and the like. When the unitdosage form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac or sugar and the like.

For administration orally, the compounds may be formulated as asustained release preparation. Numerous techniques for formulatingsustained release preparations are described in the followingreferences—U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917;5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263;6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379;5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362;5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004;5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177;and WO 98/47491. These references are hereby incorporated herein byreference in their entireties. In a preferred embodiment, the sustainedrelease formulation utilized has an enteric coating.

For administration by inhalation, the active compound(s) may beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

A syrup or elixir may contain the active agent, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive components may be incorporated into sustained-releasepreparations and devices including, but not limited to, those relying onosmotic pressures to obtain a desired release profile. Once dailyformulations for each of the active components are specificallyincluded.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation or transcutaneous delivery (forexample subcutaneously or intramuscularly), intramuscular injection or atransdermal patch. Thus, for example, the compounds may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell-known in the medical arts.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLES Example 1 Radioprotection Assay

HeLa cells were treated with 2 μg/ml of chloroquine for one hour, washedfor one hour, and irradiated at 2 or 6 Gy. Subsequently, 1000 cells wereplated and assessed for colony formation. Table 1 shows that exposure tochloroquine prior to irradiation increased cell survival by 30%. TABLE 1Average Standard Treatment Number of Colonies* Deviation 2 Gy 444 19.5Chloroquine + 2 Gy 580 21.2 6 Gy 94.6 10.6 Chloroquine + 6 Gy 129 8.6*Averages were from five individual samples.

To test the possibility that chloroquine activation of ATM may causeradioprotection, C57/BL6 mice were exposed to 8 Gy IR, a dose whichkills approximately 80% of the mice at around two weeks. Death appearsto result from hematopoietic toxicities. The day before total bodyirradiation (TBI), mice were either given an i.p. injection ofchloroquine or chloroquine was added to the drinking water (5 mice—i.p.1.75 mg/kg chloroquine; 5 mice—i.p. 3.5 mg/kg chloroquine; 5 mice —1.75mg/kg chloroquine in drinking water; 5 mice —3.5 mg/kg chloroquine indrinking water). FIG. 1 shows a Kaplan-Meier survival curve indicatingthat a dose of chloroquine prior to the TBI provided significantprotection from death. Significant protection was not observed in ATMdeficient (homozygous) transgenic mice. The experiment was reproducednumerous times and analyses of tissues indicated that the protectiveeffect was due to enhanced recovery of hematopoietic cells (bone marrow,spleen, thymus) following irradiation (FIG. 2). Injection of chloroquineprior to the TBI had no effect on the survival of mice lacking ATM genes(FIG. 3), thus indicating that radioprotection may be dependent on ATM.

Treatment with chloroquine or hydroxychloroquine also providedsignificant protection against loss of coat color in surviving mice.FIG. 10 shows three pairs of mice subject to 8 Gy total bodyirradiation. The two control mice on the left of the figure showsignificant loss of coat color. The pair in the middle which weretreated with chloroquine before exposure to total body irradiation showno significant loss of coat color. The pair on the right treated withhydroxyquinolone show an intermediate extent of protection.

Example 2 Cancer Prevention

Transgenic mice expressing the c-myc oncogene under the control of theimmunoglobulin enhancer (i.e., Eμ-myc mice) develop B-cell lymphomas andleukemias with relatively short latencies. Chloroquine was added to thedrinking water of a cohort of Eμ-myc mice and the mice were observed forthe development of B-cell malignancies. FIG. 4 demonstrates that 100% ofthe control transgenic mice developed malignancies within 100 days ofbirth while 0% of the transgenic mice on chloroquine developed tumors.After ˜120 days, half of the cohort of chloroquine-treated mice weretaken off of chloroquine and the other half were switched to receiving adose of chloroquine by i.p. injection once a week. Within ˜30 days, allof the transgenic mice taken off of the chloroquine had developed tumorswhile none of the mice receiving weekly i.p. injections developedcancer. At ˜10 months of age, these mice on weekly chloroquine remainedcancer-free and appeared healthy and normal.

The carcinogen 3-methylcholanthrene (3-MC) induces soft tissue sarcomasif injected into muscle and skin carcinomas if applied to the skin(Smart, et al. (1986) Carcinogenesis 7:1669-1675; Noguchi, et al. (1996)Proc. Natl. Acad. Sci. U.S.A 93:11798-11801; Horak, et al. (1984) Br. J.Cancer 49:637-644). This model system has been used to demonstrate thatsuperinduction of p53 after DNA damage (e.g., in a mouse carrying anextra copy of chromosomal DNA containing the p53 gene) protects micefrom the development of cancers induced by chemical carcinogentreatments (Garcia-Cao, et al. (2002) EMBO J. 21:6225-6235). Therefore,it was determined whether the protective effect observed in thesestudies could likewise be achieved by biochemically enhancing p53induction. As demonstrated herein, ATM kinase activation by chloroquinedid not induce strand breaks or induce phosphorylation of substratesthat normally get phosphorylated by ATM at the sites of DNA breaks,however, it did lead to induction and phosphorylation of p53 protein.Thus, chloroquine pre-treatment may prevent/reduce tumor developmentresulting from 3-MC injections. Accordingly, doses of 3.5 mg/kg ofchloroquine were given by i.p. injection 24 and 4 hours prior to 3-MCinjection in 30 mice. Results are shown in FIG. 5. The occurrence ofthese tumors was readily apparent by visual inspection and confirmed byhistologic assessment.

In a further experiment, doses of 3.5 mg/kg of chloroquine were given byi.p. injection 24 and 4 hours prior to 3-MC injection in 30 wild type(strain C57Bl/6) mice. The occurrence of these tumors was readilyapparent by visual inspection and confirmed by histologic assessment.Results are shown in FIG. 5. Treatment with chloroquine significantlyincreased the percentage of mice surviving tumor free (p=0.0013).

In a further experiment 3-MC was injected into the skin on the leg of amouse once a week for 4 weeks. Three genetic backgrounds were used:wild-type, ATM-null, and p53-null. One half of each cohort of micereceived 3.5 mg/kg of chlorquine (CHQ) via IP injection 24 hours and 4hours prior to each of the four 3-MC administration. The development ofskin carcinomas was followed over time. FIG. 7 shows tumor incidence inmice receiving either placebo or chloroquine prior to 3-MC injection.Chloroquine markedly protected from tumor development. FIG. 8 showstumor incidence in ATM-null mice receiving either placebo or chloroquineprior to 3-MC injection. Chloroquine does not protect from tumordevelopment. FIG. 9 shows tumor incidence in p53-null mice receivingeither placebo or chloroquine prior to 3-MC injection. Again chloroquinedid not protect from tumor development. These results showsthat that theprophylactic effect of chloroquine is mediated at least in part throughATM and p53.

Multiple exposures to non-lethal doses of ionizing radiation can inducethymic lymphomas in C57BL/6 mice (Boniver, et al. (1990) Int. J. Radiat.Biol. 57:693-698). Using a classical, tumor-inducing protocol (Kaplanand Brown (1952) J. Natl. Cancer Inst. 13:185-208), which consists offour weekly whole-body exposures of 1.75 Gy each, the effect ofchloroquine administration on thymic lymphoma formation was examined.Chloroquine (3.5 mg/kg) was administered to 4-week old female C57BL/6mice by i.p. injection 24 hours and 4 hours prior to each of the fourdoses of radiation described in the protocol. According to the protocol,tumors were expected to appear within 4-6 months after the last dose ofirradiation in 90% of control (untreated) mice. FIG. 6 shows the resultsof this analysis.

All patents, publications, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual patent, publication, or patent application wasspecifically and individually indicated to be incorporated by reference.

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made thereto without departing from thespirit or scope of the appended claims.

1. A method of treatment for a radiation related disorder comprisingadministering to an animal subject in need thereof an effective amountof a chloroquine compound.
 2. The method of claim 1 wherein saidradiation related disorder is caused by an exposure to a non-ionizingradiation.
 3. The method of claim 1 wherein said radiation relateddisorder is caused by an exposure to an ionizing radiation.
 4. Themethod of claim 3 wherein said chloroquine compound treats a skin damagesymptom caused by said ionizing radiation.
 5. The method of claim 4wherein said skin damage symptom is at least one symptom selected fromswelling, itching, redness of skin, and hair loss.
 6. The method ofclaim 3 wherein said chloroquine compound treats a non-skin damagesymptom caused by said ionizing radiation.
 7. The method of claim 6wherein said non-skin damage symptom is at least one symptom selectedfrom nausea, vomiting, diarrhea, loss of appetite, fatigue, fever,diminished organ function, seizures, cancer, and coma.
 8. The method ofclaim 1 wherein said radiation related disorder is acute radiationsyndrome.
 9. The method of claim 1 wherein said chloroquine compoundprevents a death due to radiation exposure.
 10. The method of claim 1wherein said chloroquine compound protects from sub-lethal and/orunintentional radiation exposure.
 11. The method of claim 1 wherein saidchloroquine compound protects from sub-lethal and/or lethal intentionalradiation exposure.
 12. The method of claim 11 wherein said intentionalradiation exposure is due to an act of terrorism.
 13. A method oftreatment for an adverse effect of a radiation therapy comprisingadministering to an animal subject in need thereof an effective amountof a chloroquine compound.
 14. The method of claim 13 wherein saidadverse effect is at least one effect selected from a skin damage,alopecia, or neutropenia.
 15. The method of claim 1 or 13 wherein saidchloroquine compound is at least one compound selected from chloroquine,chloroquine phosphate, hydroxychloroquine, chloroquine diphosphate,chloroquine sulphate, hydroxychloroquine sulphate, or enantiomers,derivatives, analogs, metabolites, pharmaceutically acceptable salts,and mixtures thereof.
 16. The method of claim 15 wherein said compoundis at least one compound selected from chloroquine, chloroquinephosphate, hydroxychloroquine, chloroquine diphosphate.
 17. The methodof claim 16 wherein said compound is chloroquine.
 18. The method ofclaim 16 wherein said compound is hydroxychloroquine.
 19. The method ofclaim 17 or 18 wherein said compound is an essentially pure (+) isomer.20. The method of claim 17 or 18 wherein said compound is an essentiallypure (−) isomer.
 21. The method of claim 1 or 13 wherein the amount ofthe compound administered is at least about 0.1 mg/kg/day.
 22. Themethod of claim 1 or 13 wherein the amount of the compound administeredis up to about 10 mg/kg/day.
 23. The method of claim 1 or 13 wherein theamount of the compound administered is more than about 0.1 mg/kg/day. 24The method of claim 1 or 13 wherein the amount of the compoundadministered is more than about 1.0 mg/kg/day.
 25. The method of claim 1or 13 wherein the amount of the compound administered is less than about50 mg/kg/day.
 26. The method of claim 1 or 13 wherein the amount of thecompound administered is less than about 10 mg/kg/day.
 27. The method ofclaim 1 or 13 wherein the chloroquine compound is administered more thanonce a week.
 28. The method claim 1 or 13 wherein the chloroquinecompound is administered daily, every two weeks, or once a month. 29.The method of claim 1 or 13 wherein the chloroquine compound isformulated in a sustained release formulation.
 30. The method of claim 1or 13 wherein said amount of said compound administered is about 0.1 toabout 9 mg/kg once a week.